Immunofluorescence Protocol & Fiji Analysis

General Preparation

• Temperature equilibration

  • Warm all aqueous buffers and stock solutions to $37^\circ \text{C}$ before use to avoid thermal shock to tissue sections or cells.

• Safety & waste

  • $4\%$ PFA is toxic → handle in a fume hood; collect liquid waste in a designated PFA container.

  • Work with light-sensitive fluorophores in dim light; keep tubes & slides wrapped in aluminium foil whenever the note says "in the dark".

Fixation

• Standard fixation (thin sections / cells)

  • Remove frozen sections from the freezer.

  • Immediately add ice-cold $4\%$ PFA for $20\;\text{min}$ at room temperature.

• Preparation of $4\%$ PFA from $10\%$ stock

  • Mix $20\;\text{mL}$ of $10\%$ PFA + $30\;\text{mL}$ PBS $\Rightarrow$ $50\;\text{mL}$ of working-strength $4\%$ PFA.

• Alternative fixation (large/bigger tissues)

  • Incubate overnight at $4^\circ \text{C}$ in $4\%$ PFA to allow complete penetration.

• Post-fix washes

  • Wash $3 \times$ with PBS,$15\;\text{min}$ each at room temp to remove residual fixative.

Permeabilization (conditional)

• When to use: Only if the target protein is intracellular; do NOT use for membrane-associated antigens (detergent disrupts membranes).

• Reagent: $0.1\%$ Triton X-100 in PBS.

  • Example recipe: Add $50\;\mu\text{L}$ Triton X-100 (100 \%) to $50\;\text{mL}$ PBS $\Rightarrow$ 0.1% final titration.

• Incubate slides/coverslips for $1\;\text{h}$ at $37^\circ \text{C}$.

• Wash $3 \times$ with PBS, $5\;\text{min}$ each at RT.

Blocking of Non-specific Binding

• BSA block

  • Incubate $30\;\text{min}$ at RT in $5\%$ BSA in PBS.

  • Preparation example: $2.5\;\text{mL}$ BSA (w/v stock) + $47.5\;\text{mL}$ PBS $\Rightarrow$ $50\;\text{mL}$ of $5\%$ BSA.

  • Commercial alternative: Thomas Scientific BSA Blocker 10× $(1{:}10)$ dilution → $30\;\text{min}$ room temp.

• Serum block (to quench Fc-receptors & reduce species cross-reactivity)

  • Incubate $30\;\text{min}$ at RT with $5\%$ normal serum from the species in which the secondary antibody (2°) was raised (e.g., goat serum for goat-derived 2° Abs).

• PBST option

  • For highly hydrophobic epitopes: $1\%$ BSA in PBST (PBS + $0.05{-}0.1\%$ Tween-20).

Primary Antibody Incubation

• Dilute primary antibody in $1\%$ BSA in PBST.

• Incubate overnight (≈ $16\;\text{h}$ ) at $4^\circ \text{C}$ in a humidified dark chamber to prevent evaporation & photobleaching.

Post-Primary Washes

• Remove primary solution.

• Wash $3 \times$ with PBS, $5\;\text{min}$ each, gentle rocking, in the dark.

Secondary Antibody Incubation

• Dilute fluorophore-conjugated 2° antibody in $1\%$ BSA (same buffer as primary).

• Incubate $1\;\text{h}$ at RT, in the dark.

• Wash $3 \times$ with PBS, $5\;\text{min}$ each, in the dark.

Mounting & Curing

• Add a small drop of mounting medium containing DAPI (e.g., FluoroShield with DAPI) to the slide.

• Invert the coverslip onto the drop, avoid bubbles.

• Allow to cure $24\;\text{h}$ at $4^\circ \text{C}$ in the dark.

• Store slides at $4^\circ \text{C}$; protect from light to preserve fluorescence.

Antibody Reference Table

• Primary (1°) antibodies

  • anti-PLK1: Abcam #ab17057, host = mouse, dilution $1{:}500$.

  • anti-phospho-PLK1 (p-PLK1): Abcam #ab12157, host = rabbit, dilution $1{:}200$.

• Secondary (2°) antibodies

  • Alexa Fluor-conjugated anti-mouse IgG: Invitrogen #A28175, dilution $1{:}1000$.

  • Alexa Fluor-conjugated anti-rabbit IgG: Abcam #ab150077, dilution $1{:}500$.

• Manufacturer protocol reference: https://www.abcam.com/protocols/immunocytochemistry-immunofluorescence-protocol

Brief Rationale / Conceptual Connections

• Fixation cross-links proteins (PFA → methylene bridges) to preserve morphology.

• Permeabilisation with Triton X-100 punches nanometer-sized holes in the lipid bilayer for antibody access.

• Blocking agents (BSA/serum) occupy free aldehydes & non-specific binding sites → lower background.

• Primary → Secondary system amplifies signal: multiple 2° Abs bind each 1° Ab, each 2° carries several fluorophores.

• Temperature & dark conditions prevent fluorophore bleaching and maintain protein conformation.

Ethical / Practical Considerations

• Dispose of PFA according to institutional chemical‐hazard rules.

• Use appropriate ethical sourcing for animal-derived sera; document lot numbers for reproducibility.

ImageJ / Fiji – Nuclear Quantification Workflow (DAPI channel)

1. Open image: File → Open (load .dapi file).

2. Split channels: Image → Color → Split Channels → Keep the blue channel; close red & green to minimise RAM usage.

3. Threshold: Image → Adjust → Threshold.

   • Adjust sliders until background = white, nuclei = black.

   • Confirm by checking that no true nuclei are excluded and minimal background is included.

4. Binarise: Process → Binary → Make Binary.

5. Fill Holes (solidify nuclei): Process → Binary → Fill Holes.

6. Watershed (separate touching nuclei): Process → Binary → Watershed.

7. Particle analysis: Analyze → Analyze Particles.

   • Set size 30-infinity, check pixel units

   • Circularity (e.g., $0.00{-}1.00$ ) to exclude debris.

   • Show Outlines

   • Output summarize and include holes.

Common Pitfalls & Tips

• Incomplete fixation → antigen loss; ensure at least $20\;\text{min}$ (thin) or overnight (thick) exposure.

• Over-permeabilisation destroys membrane epitopes; skip Triton step for transmembrane proteins.

• Fluorophore bleaching: always work on ice / in the dark once fluorophores are present.

• During Fiji thresholding, use Histogram → Check ‘dark background’ if needed.

Quick Timing Overview (thin section example)

• Fixation: $20\;\text{min}$

• Washes: $3\times15\;\text{min}$ ($\approx$ $45\;\text{min}$ )

• Permeabilisation: $60\;\text{min}$

• Blocking: $30\;\text{min}$ + $30\;\text{min}$ (serum)

• Primary: $\sim$ $16\;\text{h}$ (overnight)

• Secondary: $60\;\text{min}$

• Mount & cure: $24\;\text{h}$

Total hands-on time ≈ $3\;\text{h}$ spread over two days (+ overnight primary, + $24\;\text{h}$ curing).